SUMMARY FTY720 is FDA-approved oral treatment for multiple sclerosis (MS). At the dose used to treat MS, FTY720's effects on sphingosine-1-phosphate receptors result in the sequestration of lymphocytes in secondary lymphoid organs. At much higher doses, many groups have demonstrated that FTY720 is a selective and effective anti-neoplastic agent that works against a wide variety of tumor types. In mouse cancer models at 5-10 mg/kg, FTY720 inhibits the growth of both primary tumors and metastases while exhibiting minimal toxicity. Despite this striking success in animal models, FTY720 cannot be tested in human cancer patients because it triggers profound bradycardia by activating S1P receptors 1 and 3 before the anti-neoplastic dose is reached. However, we have recently demonstrated that the anti-neoplastic effect of FTY720 occurs through an S1P receptor-independent mechanism that includes nutrient transporter down-regulation. While FTY720 analogs that fail to activate S1P receptors have promising anti-neoplastic activity and therapeutic potential, the molecular target of FTY720 in cancer models remains unknown. In this proposal, we will use a chemical biology approach to identify the target of FTY720 and its anti-neoplastic analogs in cancer cells. This project has two Specific Aims: 1) Generate photo-affinity labeling probes from FTY720 analogs that are active in intact cells, and 2) Isolate proteins that specifically interact with the probes using a series of negative controls to identify non-specific binding partners. These probes will include a diazirine to allow UV-crosslinking to the target and an alkyne to covalently couple a biotin to the probe-target complex via click chemistry. Determining the molecular target for FTY720 and its analogs in cancer models is a critical step in the development of this novel approach to cancer therapy because it will allow us to: 1) define the molecular characteristics of tumors that will be susceptible to this therapeutic approach so that drugs can be tested in the correct patient populations, 2) uncover potential mechanisms of resistance to these drugs, 3) anticipate potential toxicities, 4) make predictions about drug combinations that are likely to be synergistic, and 5) test our model for how these compounds selectively kill cancer cells using standard molecular and biochemical techniques.